Blockade of ROCK inhibits migration of human primary keratinocytes and malignant epithelial skin cells by regulating actomyosin contractility.

Actomyosin contractility, crucial for several physiological processes including migration, is controlled by the phosphorylation of myosin light chain (MLC). Rho-associated protein kinase (ROCK) and Myosin light chain kinase (MLCK) are predominant kinases that phosphorylate MLC. However, the distinct roles of these kinases in regulating actomyosin contractility and their subsequent impact on the migration of healthy and malignant skin cells is poorly understood. We observed that blockade of ROCK in healthy primary keratinocytes (HPKs) and epidermal carcinoma cell line (A-431 cells) resulted in loss of migration, contractility, focal adhesions, stress fibres, and changes in morphology due to reduction in phosphorylated MLC levels. In contrast, blockade of MLCK reduced migration, contractile dynamics, focal adhesions and phosphorylated MLC levels of HPKs alone and had no effect on A-431 cells due to the negligible MLCK expression. Using genetically modified A-431 cells expressing phosphomimetic mutant of p-MLC, we show that ROCK dependent phosphorylated MLC controls the migration, focal adhesion, stress fibre organization and the morphology of the cells. In conclusion, our data indicate that ROCK is the major kinase of MLC phosphorylation in both HPKs and A-431 cells, and regulates the contractility and migration of healthy as well as malignant skin epithelial cells.